JP2007203034A - Member for prosthesis and culture of organic tissue or release of medicine in body and method for manufacturing the member - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a transplanting process for a ceramics corpusculum by utilizing gelation reaction of salt alginate with salt having bivalent cation. <P>SOLUTION: A reactant of salt having bivalent cation adhered on a surface of ceramics corpusculum with a solution of salt alginate is provided between ceramics corpuscula to prevent leakage and moving from a transplanted section. It acts effectively in prosthesis for a hard tissue in an organic hard tissue loss section as a temporary prosthetic object as bone filler and can be easily manufactured. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a prosthetic material used as a bone filler for restoring bone tissue.

  Conventionally, bone-shaped fillers of block shape and crushed type indefinite shape mainly composed of calcium phosphate have been widely applied. JP-A-10-158075 and JP-A-2003-335574 disclose a method for producing a spherical ceramic, and devise to improve the filling rate and operability in a bone defect part.

However, the block-shaped bone filler has a certain standard shape, and is difficult to adhere to a defect part having a different shape in each case. Although the crushing type indefinite shape has excellent operability for filling the defect portion, the filling density at the transplantation site becomes high, so that the blood flow is prevented from entering the transplantation site which is supposed to promote bone regeneration. In addition, in the initial stage of transplantation, the fixation of the transplant material does not proceed and leakage may occur. In the spherical shape, since the spheres are embedded in a point contact state, the above-mentioned filling density is reduced, and it is considered that the void into which the bloodstream enters can be secured and work effectively for bone regeneration. Produce.

In St. Marianna University School of Medicine, Vol. 28, pages 689-697, 2000, as a method for preventing the leakage of bone filler, crush-type amorphous β-TCP ceramics and fibrin glue, a biological tissue adhesive, were used. Mixing usage is being considered. However, voids between bone fillers are filled, and the new bone formation is delayed due to the presence of fibrin glue, and since fibrin itself is an animal-derived biomaterial, it may cause infection when applied to humans. . In addition, adjustment of fibrin glue requires complicated procedures.

In JP-A-2004-201799, there is a method for producing a gel-like transplant that is easily filled by mixing hydroxyapatite granules, sodium alginate, and platelet-rich plasma collected from the patient himself as a bone filler in the oral cavity. It is shown. However, collecting platelet-rich plasma requires blood collection from a patient and complicated procedures, and the volume that can be used is limited, so that it is difficult to apply to large bone defects.
Japanese Patent Laid-Open No. 10-158075 JP 2003-335574 A JP 2004-201799 A JP 63-226360 A St. Marianna University School of Medicine, Vol. 28, pages 689-697, 2000

Problems to be solved include leakage and movement of the prosthetic material until the wound surface is closed when using the ceramic body as a prosthetic material, and maintaining point contact when the ceramic body is a spherical bone filler In other words, the disease may be caused by infectious diseases that may occur when a prosthetic material is fixed by a method using an animal-derived biomaterial, securing a void into which blood flow enters.

The present invention utilizes calcium alginate gel resulting from the reaction between alginate and a salt having a divalent cation. When ceramic bodies are used as a prosthetic material for a living body, leakage and migration from the implantation site are prevented. In particular, when the ceramic body is a spherical bone filler, the method of injecting with a syringe while maintaining the state of point contact, or the method of covering the surface of the transplant site with a gel, can be easily applied. Making the transplant possible is the main feature.
In addition to a hard tissue filling material used for prosthesis in a living body defect part, the present invention can be used as a temporary prosthesis, a biohard tissue streak, or a groove-like wound repair material for implanting artificial tooth roots and the like. When entering, it can be used as a material for temporarily fixing the excess defect portion of the jawbone that is generated.

The ceramic body in the present invention is a body such as a granule, a small block, a pulverized body, etc., and one dimension is 50 μm to 5 mm, preferably 500 to 850 μm, and each may be uniform or non-uniform. A collection of shaped bodies is illustrated. In addition, the spherical shape is preferable in terms of manufacturing, adjustment of strength, filling rate, reproducibility, operability and the like.
In addition, it is more preferable that the gap between the ceramic bodies is 50 μ to 350 μ, preferably 100 μ to 300 μ, so that a state in which bone easily enters is formed. The diameter of the ceramic body is also selected. For the gap shown here, a value obtained by calculation in a state where the ceramic body is close to a true sphere may be sufficient.
Specifically, as ceramics, hydroxyapatite, carbonate apatite, fluorapatite, chlorapatite, β-TCP, α-TCP, calcium metaphosphate, tetracalcium phosphate, calcium hydrogen phosphate, dicalcium phosphate, octacalcium phosphate, Examples include calcium hydrogen phosphate dihydrate, calcium phosphate glass, a mixture of these calcium phosphates, alumina, zirconia, and the like.
When the ceramic body is spherical, it may be porous or dense, and its production method is exemplified by a spray drying method, a rolling granulation method, etc. A technique for dripping a slurry of a ceramic precursor into a cooling solvent such as liquid nitrogen described in JP-A-10-108575, which is a technique capable of producing large spherical particles, and freeze-drying is shown. is not.
Examples of the salt having a divalent cation include alkaline earth metals, but are not limited thereto, and more specifically, chloride, lactate, acetate, gluconate, glycerophosphate, Maleate, sulfate, hydrogen orthophosphate, calcium salt, or strontium salt are exemplified, and among them, calcium salt is preferable in that it has good curability associated with gelation and provides strength.

As a method for attaching a salt having a divalent cation, for example, the above-mentioned ceramic body is converted into calcium chloride, calcium lactate, calcium acetate, calcium gluconate, calcium glycerophosphate, calcium maleate, calcium sulfate, or calcium hydrogen orthophosphate. A method of dipping in a salt solution having a divalent cation such as 10 seconds to 10 minutes and then drying is shown.

Examples of the alginate solution include sodium alginate, potassium alginate, magnesium alginate, ammonium alginate, or propylene glycol alginate solution.
The reaction method of the ceramic body to which a salt having a divalent cation is attached and the alginate solution are not particularly limited as long as they are brought into contact with each other. For example, the ceramic body is inserted. Examples include agitation and agitation, pressure molding, etc., in which the alginate solution is placed in a container, but the alginate solution and the ceramic body to which a salt having a divalent cation is attached are quickly bonded. The production is preferably carried out in an earlier form, and a method of molding with an extrusion pressure type that allows easy removal of excess alginate solution is preferred.
In the present invention, the obtained alginic acid and the salt having a divalent cation are gelled and the solidified member is freeze-dried to obtain a dried calcium alginate, which is normal and long. The period can be saved. By pouring water, physiological saline or the like again from the rear part of the syringe and pressurizing with a piston, the prosthetic material in a gelled state can be obtained again. In addition, a lyophilized prosthetic material imparted with a sustained drug release effect can be obtained by previously mixing a drug or protein in an alginate aqueous solution and freeze-drying.
The drug or protein in the present invention is, for example,
Cell growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), hepatocyte growth factor (HGF), transforming growth factor (TGF) Anti-cancer drugs such as bone morphogenetic protein (BMP), platelet-rich plasma (PRP) interferon, interleukin-2, ifosfamide, including many cell growth factors widely used in dentistry, streptomycin, gentamicin, gatifloxacin Antibiotics such as atorvastatin, pravastatin, cholesterol-lowering agents such as simvastatin, other, lidocaine, protamine sulfate, sodium iodohyproate, iodosulfoprophthalein, heparin sodium injection, glucose Note, norepinephrine note, dextran note, Examples include sodium thiopental, sodium chromate injection, xylitol, procaine hydrochloride injection, tetracaine hydrochloride, tubocurarine chloride, squismethonium chloride, amorphous insulin zinc water suspension, amyl nitrite, ajmaline, and the like.

  It is possible to prevent leakage from the transplant site when the ceramic body is used as a prosthetic material for a living body. In the case where the ceramic body is a spherical bone filler, a void due to point contact between the ceramics is secured to maintain a state in which blood flow easily enters and promote bone regeneration. Alginate compounds are components extracted from seaweed, and sodium alginate is used as a pharmaceutical product. Therefore, there is no risk of infectious diseases that are concerned with animal-derived biological materials. Further, since the gelation reaction between the alginate and the salt having a divalent cation occurs in a very short time, the operation technique is simple.

A 0.5 to 10% calcium chloride aqueous solution is prepared, and spherical ceramic bodies are immersed in the solution to evaporate only moisture. In this method, the spherical ceramics subjected to the above treatment are reacted with a 0.5 to 10% aqueous sodium alginate solution to cause gelation, and the spherical ceramics are transplanted while maintaining point contact without causing leakage. There are a molding method in which gelation is performed in a syringe and injection is possible as it is, and a method in which a sodium alginate aqueous solution is supplied from the upper part of the molding into a defect having a complicated shape and is fixed by a gel film.
The spherical ceramics described above are hydroxyapatite, carbonate apatite, fluorapatite, chlorapatite, β-TCP, α-TCP, calcium metaphosphate, tetracalcium phosphate, calcium hydrogen phosphate, dicalcium phosphate, octacalcium phosphate, Calcium hydrogen phosphate dihydrate, calcium phosphate glass, a mixture of these calcium phosphates, alumina, zirconia and the like.
In addition, the present invention may be used as a fast-curing temporary prosthetic material used during and after surgery, in addition to the bone filler filled into the bone defect.
For example, if excessive damage to the jaw bone during surgery related to dental implant placement or other hard tissues, or if emergency treatment is required, after supplementing with ceramic material with calcium ions attached, A simple treatment is possible only by dropping alginate.
The present invention provides a simple production method and a base material produced by adding a drug, a cell, etc. to an alginate solution while having a strength sufficient for prosthesis to a living hard tissue. It is also highly useful as a base material for living bodies and a biologically embedded base material for DDS.

A spherical ceramic body made of β-TCP is prepared by mixing a β-TCP powder obtained by a wet synthesis method with a 3% polyvinyl alcohol aqueous solution to prepare a slurry, which is dropped on liquid nitrogen, freeze-dried, and 1100 ° C. It was sintered for 10 hours, and a diameter of 500 to 700 μm was selected with a sieve.
Spherical ceramics (Fig. 1) made of β-TCP with a diameter of 500-700μm is immersed in a 1% calcium chloride aqueous solution for 30 seconds in a beaker, and water is evaporated in a 60 degree drier. It is loaded and covered with a thin sheet material 2 such as Parafilm (registered trademark) so that the bead-like ceramic material 3 does not leak and a gap is formed to allow the sodium alginate aqueous solution to pass therethrough. FIG. 2 shows a sheet material 2 in a state where the distal end portion of the syringe 1 whose front end portion has been cut is covered, and a ceramic material 3 having calcium ions attached thereto is placed therein, and the piston 4 is placed in the opening portion of the syringe 1. FIG.
A 1% sodium alginate aqueous solution is poured from the piston 4 side, and only the sodium alginate aqueous solution is pushed out so that the spherical ceramic material 3 does not leak from the tip. At that time, the sheet material 2 is suppressed by using a means such as holding by hand or fixing with a fixing band or the like so that the sheet material 2 is not pushed out together. After the sheet material 2 is removed, the spherical ceramic is extruded by the piston 4 to obtain the spherical ceramic in a state of being formed into a cylindrical shape.

The operation of the molding tool shown in this embodiment is shown in more detail in FIG.
Reference numeral 10 denotes a cylindrical body, for example, a syringe with the above-described tip cut. Reference numeral 11 denotes a lid, which forms a rotating portion 11a that is rotatably connected to a part of the cylindrical body 10, is rotatably mounted, and forms a locking portion 11b at an opposing portion thereof.
In a state where the engaging portion 11b of the lid portion 11 is engaged with the concave portion 10b of the cylindrical body 10, spherical ceramic particles 13 treated with a salt having a divalent cation are placed inside (FIG. 3 (a)). ).
Further, a sodium alginate solution 15 is injected and pushed by the piston 14.
The solution 15 which has moved the piston 14 and passed between the pressed ceramic particles 13 is discharged to the outside through the hole 12 provided in the lid 11.
When the sodium alginate solution 15 is completely released by the movement of the piston 14,
The locking portions 10b and 11b of the lid portion 11 are unlocked, the lid portion 11 is rotated around the movable portion 11a, and the press-molded ceramic 16 is taken out.
The spherical ceramic particles treated with a salt having a divalent cation can be easily molded and manufactured as a ceramic prosthesis material by pressurization and a very rapid gelation reaction of sodium alginate.

The prosthetic material made of the spherical ceramics and calcium alginate prepared in Example 1 was put in physiological saline, and the form after 72 hours was observed to verify the degradability of alginic acid. As a result, it was confirmed that the alginic acid gel was dissolved and the shape was slightly broken even after 72 hours, but the tubular shape was maintained.

The prosthetic material made of spherical ceramics and calcium alginate prepared in Example 1 was examined as to whether or not a body fluid such as blood would infiltrate by immersing the red ink for writing into the blood. As a result, it was confirmed that both the gel and the spherical ceramic were stained red, and blood infiltration from the outside was possible.

When the spherical ceramic obtained in Example 1 was filled into a hole in which the defect portion was supposed and a 1% sodium alginate aqueous solution was dropped from above the hole, a gel film was formed to close the hole, and the spherical ceramic was fixed. Due to the presence of this gel film, leakage of spherical ceramics was prevented and the position could be fixed.
According to the embodiment, if the spherical ceramic particles subjected to calcium salt treatment are filled in the defect portion and supplied to the surface by dropping the alginate solution, the dripped range is instantly cured. In addition, it can be used for temporary prostheses, and can be used as a so-called instantaneous fixing agent.

FIG. 4 is a diagram showing an example of an instrument for producing a prosthetic material and the like used in the embodiment of the present invention.
In the embodiment shown in FIG. 4, the lid portion shown in FIG. 3 is an accumulation type, and excess sodium alginate is discharged through one through hole 22.
After the lid portion 21 is accumulated and fixed to the cylindrical body 20 and filled with a plurality of spherical ceramic beads 23 subjected to calcium coating treatment (a), for the purpose of cell culture, an alginate solution 25 containing cells is added. In the case of sustained drug release, an alginic acid solution 25 containing a target drug is injected and pressed with the pressing tool 24 forming the pressing surface 241 (20a).
As the alginic acid solution 25 flows through the gaps between the beads, the excess solution reaches the lid 21 and the holes 22 are released (20c) and (b).
Furthermore, after pressing the pressing tool 24 to such an extent that the cells are not destroyed, the alginic acid solution and the calcium salt on the bead surface are gelled and solidified 26 as a whole to turn the lid 21 to release the accumulation (20d). (C).
Further pressing (20e), the solid material 26 is taken out (d).

Sustained release of drugs

A 2% sodium alginate aqueous solution was prepared with an aqueous solution containing basic fibroblast growth factor (bFGF) at 1 μg / ml, and 0.2 ml was added from the back of the syringe to obtain a prosthetic material composed of spherical ceramics and calcium alginate. . This was transferred into a 24-well plate containing 2 ml of Hank's solution and allowed to stand in a 37 ° C. incubator.
The supernatant was collected on the 1st, 4th, and 7th, and bFGF released into Hanks' solution was quantified by ELISA. The results are shown in FIG. As a result, the release amount of bFGF contained in calcium alginate increased as the standing time increased. From these results, it was confirmed that the present invention can be applied as a bone filler having the property of gradually releasing growth factors, drugs and the like that promote bone formation consisting of proteins.

Cell culture

A 2% sodium alginate aqueous solution containing human mesenchymal stem cells at a concentration of 250,000 cells / ml was prepared, and 0.2 ml was poured from the back of the syringe to obtain a ceramic composite. This complex was cultured for 14 days in a bone differentiation medium containing β-Glycerophosphate, L-ascorbic acid phosphate magnesium salt hydrate, and dexamethasone, and the surface was scanned with electrons. As a result, it was observed that osteoblast-like cells were attached to the surface of the calcium alginate gel (FIG. 6).
Moreover, when only cells were collected from the prosthetic materials cultured on the 7th and 14th, and the expression of alkaline phosphatase, which is an index of bone differentiation, was measured by the real-time PCR method, the expression was increased (FIG. 7). ). From this result, it was shown that this prosthetic material can be applied to bone regenerative medicine as a three-dimensional culture carrier for mesenchymal stem cells.

Animal experimentation

A prosthetic material made of spherical ceramics and calcium alginate obtained based on Example 1 was transplanted subcutaneously into nude mice, and 8 weeks after transplantation, the prosthetic material was taken out from the transplanted site to prepare a tissue section. As a result, an observation image in which bones were formed between the spherical ceramics as shown in FIG. 8 was obtained. From this result, it was shown that it can be used as a bone filler with bone guidance.

  Use as a prosthetic material in orthopedics and dentistry, especially as a bone filling material used for bone defects, as well as sustained release of drugs used by embedding three-dimensional cell culture substrates, bones, and subcutaneouss It can be applied to a carrier (base material).

The figure which shows the scanning microscope picture of the spherical ceramics used by this invention. The figure for demonstrating the Example of this invention. The figure for demonstrating the Example of this invention. The figure for demonstrating the other Example of this invention. The figure for demonstrating the Example of this invention. The figure for demonstrating the Example of this invention. The figure for demonstrating the Example of this invention. The figure for demonstrating the Example of this invention.

Explanation of symbols

1 Syringe 2 Sheet material 3 Ceramic material 4 Piston

Claims (14)

  A prosthetic material obtained by interposing a reaction product of a salt having a divalent cation attached to the surface of a ceramic body and an alginate solution between the ceramic bodies. A method for producing a prosthetic material, wherein the surface of a ceramic body is treated with a salt having a divalent cation and then reacted with an alginate solution.   A cell culture substrate in which a reaction product of a salt having a divalent cation attached to the surface of a ceramic body and an alginate solution is interposed between the ceramic bodies. A method for producing a cell culture substrate comprising treating a surface of a ceramic body with a salt having a divalent cation and then reacting with a salt of an alginate solution.   The cell culture substrate and the method for producing the same according to claims 3 and 4, wherein cultured cells are contained in the alginate solution.   A sustained-release drug base material comprising a reaction product of a salt having a divalent cation adhered to the surface of a ceramic body and an alginate solution interposed between the ceramic bodies. A method for producing a sustained-release drug substrate, comprising treating a surface of a ceramic body with a salt having a divalent cation and then reacting with a salt of an alginate solution.
The drug sustained-release base material and the method for producing the same according to claim 5 and 6, wherein the drug or protein is contained in an alginate solution.   After filling the body with a ceramic body that has been treated with a salt having a divalent cation, a prosthetic material, cell culture substrate or drug sustained-release substrate with an alginate solution applied to the surface of the filling site Forming method. After filling the ceramic body that has been treated with a salt having a divalent cation, it is filled with an alginate solution, pressurized from the direction of the alginate solution, and pushed out of the alginate solution to push out the divalent cation. A prosthetic material, a cell culture substrate or a drug sustained-release substrate according to claim 2, 4 and 7, wherein a molded product is obtained by interposing a reaction product of a salt having a salt and an alginate between the ceramic bodies. Method.   The ceramic is hydroxyapatite, carbonate apatite, fluorapatite, chlorapatite, β-TCP, α-TCP, calcium metaphosphate, tetracalcium phosphate, calcium hydrogen phosphate, dicalcium phosphate, octacalcium phosphate, calcium hydrogen phosphate A hydrate, a calcium phosphate glass, a mixture of these calcium phosphates, alumina, zirconia, and the like. 11. A biological tissue prosthetic material, a cell culture substrate or a drug sustained-release substrate, and a method for producing the same . The salt having a divalent cation is chloride, lactate, acetate, gluconate, glycerophosphate, maleate, sulfate, hydrogen orthophosphate, calcium salt, or strontium salt. A member for performing prosthesis or culture of the biological tissue according to 1 to 10, and a method for producing the member. The member for performing prosthesis or culture of biological tissue according to claim 1 or 10, wherein the alginate solution is a sodium alginate, potassium alginate, magnesium alginate, ammonium alginate, or propylene glycol alginate solution. . The member for performing prosthesis or culture of biological tissue according to claim 1 or 10, wherein the ceramic body is a spherical particle having a system in which the gap between the bodies is about 100 µm to 300 mm. how to.

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